development of diagnostic enzyme assay to detect leuser virus

Leuser virus

1. Development of diagnostic enzyme assay to detect Leuser virus By Naz

2. Introduction Leuser Virus also known as the yellow eye disease. It has been emerging in South-East Asia in the past few months.. 2 emerging strains, strain 1 (wild type), strain 2 (lethal) Symptoms: yellow eye, mild flu-like symptoms however if untreated it can cause a sudden rapid decline and death. Diagnostic ELISA-based blood test. ELISA is very expensive and pick up both strains of the virus, so it can’t distinguish between the two strains. The aim of this report is to develop a yellow-eye diagnostic assay based on enzyme X, and to whether the assay is a good candidate for full clinical evaluation . - Optimisation of assay (pH, temperature, wavelength, incubation time). - Determination of clinical cut-offs for the assay, to distinguish patients infected with strain 2.

3. Materials and Method Optimisation of assay PH-10 Temp-37 o C Absorbance 420nm Determination of Cut-offs (Set A & Set B) 100ml of assay buffer was prepared at a PH of 10 1ml of substrate was added to each test tube. Tubes were placed in the water bath at (37 o C) for 5 minutes, then 0.1ml of patient serum samples were added to each tube for 15 mins. After 15 mins 1ml of sodium hydroxide was added to each tube to stop the reaction. The absorbance was read at 420nm.

4. Results Figure 1- Figure Showing the concentration of enzyme X against the absorbance. Known concentration (mg/L) Measured concentration (mg/L) QC1 5 0.620 QC2 2.57 0.342 Quality control measured concentration (mg/L)

5. Results Figure 2- Figure showing the standard curve for patient set A against the absorbance. Cut-off concentration =0.4

6. Results 2 X 2 table for cut-off 0.4 Figure 3- Figure showing the standard curve for patient set B against the absorbance. Bellow the cut-off shows strain 1 (mild) and above the cut-off shows strain 2 (lethal). Cut-off concentration = 0.4 Strain 1 Strain 2 Positive 21(true +) 2 (false +) Negative 2 (false - ) 15 (true - ) False positives = 2 False negatives = 2

7. Clinical sensitivity and specificity was calculated: 91% sensitivity- The proportion of patients with the strain 1 were correctly identified by th e test. 88 % specificity- The proportion of patients without strain 1 (meaning strain 2) were correctly identified by the test. Post Test Likelihood PPV 91% NPV 88% Table 2. Post Test Likelihood– Positive and negative predictive values PPV- 91% likelihood of patients having strain 1. NPV- 88% likelihood of patients not strain 1 Lower NPV as there was a lower specificity value

8. Conclusion Low number of false negatives (2) = High sensitivity Low number of false positives (2) = High specificity Clinical sensitivity (91%) and clinical specificity (88%) confirmed and validated enzyme assay reliability. These results have proven to be considered for future investigations and relevant for the development of enzyme X assay-based diagnostic for detection of leucer virus. Limitations Low QC values- enzyme X assay was not accurate - triplicates, to limit the freeze-thaw cycles. .